The goal is to elucidate at the molecular level the factors involved in the development, function, and regulation of muscarinic acetylcholine receptors (mAChR) in the heart. We will study chick cardiac mAChR during embryonic development in vivo and in cardiac muscle cell culture. We will determine the molecular basis of the diminished physiological responsiveness of newly synthesized mAChR to confirm our hypothesis that the mAChR is synthesized in an immature form and is subsequently converted to a more physiologically active form. We have also obtained preliminary evidence by quantitative immunoblotting that at least 2 of the subunits of the GTP regulatory proteins (N Alpha and Beta) are increased greatly in amount in embryonic atria but not ventricles between days 10 and 15. It has been reported that an atrial-specific increase in mAChR number which is dependent on innervation also occurs at this time. This proposal will test the hypothesis that the mAChR and its GTP-binding effector proteins are coordinately regulated during embryonic development by innervation. We have obtained preliminary evidence that treatment of cultured cardiac cells with phorbol esters (which activate protein kinase C) prevents the mAChR-mediated increase in K+ permeability. We will test the hypothesis that short-term desensitization of the mAChR-induced negative chronotropic response is mediated by protein kinase C. We will use two different methods to isolate antibodies specific for the mAChR: by immunizations with purified mAChR preparations, and by immunization with several monoclonal antibodies specific for mAChR ligands to isolate anti-idiotype antibodies which recognize the ligand binding site on the mAChR. These antibodies will be used in our subsequent studies on mAChR regulation. This research should identify the factors involved in cardiac mAChR regulation, development, and function, and may aid in the understanding of a number of cardiac abnormalities.